A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on CDROM.
1. Field of the Invention
The present invention generally relates to the field of plants. More particularly, the present invention involves plant responses to stress and methods of altering these responses. Still more particularly, the present invention involves transgenic plants which have altered expression of phospholipase D which thereby affects plant transpiration, respiration, and bioremediation. Finally, the present invention involves breeding and selecting such transgenic plants for growth in stress-prone areas.
2. Description of the Prior Art
Terrestrial plants lose water primarily via stomata, which are pores defined by pairs of guard cells. These guard cells and stomata are located throughout the epidermis of plant stems and leaves. When subjected to heat and light, each pair of guard cells separates, thereby forming the stomata therebetween wherein plant transpiration and respiration occur. During respiration, when the stomata are open, carbon dioxide and oxygen enter and exit the leaf. When carbon dioxide enters, it participates in photosynthesis and releases oxygen as a waste product. The oxygen then passes out of the leaf through the open stomata. Additionally, oxygen also enters the leaf and takes part in respiration, thereby forming carbon dioxide as a waste product. This carbon dioxide exits the leaf via the stomata.
During transpiration, water, in the form of vapor, exits the stomata. It has been determined that more than 90% of the water loss in terrestrial plants is through the stomata. Plants minimize water loss and evaporation through the stomata in a number of ways. For example, more stomata are located on the underside of a leaf (the side of the leaf which faces the ground) than on the upper side. Stomata also close at night in response to a decreased amount of light, thereby increasing water conservation. Stomata also close in response to decreasing amounts of available water. This stomatal closure is crucial for maintaining hydration status in leaves and therefore contributes to plant survival during times of drought.
Phospholipase D (PLD) hydrolyzes phospholipids, generating phosphatidic acid (PA) and free head groups. This enzyme has been implicated in various processes, including signal transduction, membrane trafficking, cytoskeletal rearrangement, and membrane degradation. Suppression of a PLD in Arabidopsis has been shown to decrease the rate of abscisic acid (ABA)-promoted senescence in detached leaves. Other experiments have shown that the addition of phosphatidic acid (PA), a potential PLD reaction product, to protoplasts of barley aleurone and Vicia faba guard cells partially mimicked the effect of ABA. Activity and gene expression of PLD also increased in tissues treated with ABA and in plants under water deficit. (Xu, et al. Promoter Analysis and Expression of a Phospholipase D Gene from Castor Bean, 115 Plant Physiol 387-395 (1997); Jacob, et al. Abscisic Acid Signal Transduction in Guard Cells is Mediated By Phospholipase D Activity, 96 PNAS 12192-12197 (1999); and Frank, et al. Water Deficit Triggers Phospholipase D Activity in the Resurrection Plant Craterostigma plantagineum, 12 The Plant Cell 111-123 (2000)).
Because the physiological role of PLD in plants has not been established, the increases in PLD activities and gene expression shown in those studies provide no direct evidence for a role of PLD in plant response to ABA or water deficit. In addition, multiple forms of PLD in plants have been identified recently, and they exhibit different biochemical properties and patterns of expression. This raises a question of which PLD is involved in guard cell regulation. Moreover, the process which promotes stomatal closure during periods of drought stress has not heretofore been determined. Selective regulation and modification of stomatal closure would contribute to the development of drought resistant plants, plants with modified rates of respiration, transpiration, and bioremediation, and plants which react to drought stress in a quicker, more efficient manner.
The present invention overcomes the problems of the prior art and provides a distinct advance in the state of the art by providing methods of altering drought response in plants, genetically altered plants which have modified stomatal responses in comparison to wild-type plants, methods of selecting for plants having upregulated or down regulated stomatal closure, methods of testing plants for stomatal closure, and methods of differentiating between wild-type plants and plants which have been genetically altered according to the present invention.
It has now been determined that the hormone abscisic acid (ABA) promotes stomatal closure and that phospholipase D (PLD) participates in the regulation of stomatal closure induced by ABA and water stress. Three distinct PLDs, PLDxcex1, PLDxcex2 and PLDxcex3, have been cloned from Arabidopsis (Dyer et al., 109 Plant Physiol 1497 (1995); Pappan et al., 272 J. Biol. Chem. 7048-7054 (1997); Qin et al., 272 J. Biol. Chem. 28267-28273 (1997)). PLDxcex1 is expressed in Arabidopsis guard cells, and the introduction of a PLDxcex1 antisense gene abrogated its expression. The sequence of the PLDxcex1 antisense gene is provided herein as Sequence ID No. 1. Preferably, sequences having at least about 60% sequence similarity or 50% sequence identity with SEQ ID No. 1 are introduced into the PLD genome and suppress expression of PLDxcex1. More preferably, such sequences have at least about 70% sequence similarity or 65% sequence identity with SEQ ID No. 1. Most preferably, such sequences have at least about 90% sequence similarity or 85% sequence identity with SEQ ID No. 1. Plants expressing decreased amounts of PLDxcex1 also exhibit a decreased sensitivity to ABA as well as impaired stomatal closure. PLDxcex1-depleted plants exhibited an accelerated rate of transpirational water loss and decreased ability to tolerate drought stress. Overexpression of PLDxcex1 increased the leaf""s sensitivity to ABA in promoting stomatal closure and decreased the rate of transpirational water loss. Thus, PLD plays a crucial role in controlling stomatal movement and the plant""s tolerance to water deficit.
To investigate the function of PLD in plant-water relations, the presence of PLDxcex1 in Arabidopsis guard cells was determined using immunolabeling with isoform-specific antibodies raised against PLDxcex1. To perform this testing, Arabidopsis plants were grown. After 4-5 weeks of growth, fully expanded Arabidopsis leaves were detached. Epidermal peels were collected from the abaxial side of Arabidopsis leaves immediately following detachments and incubated for 1 hour in a solution containing 5 mM MES-KOH (pH 6.1), 22 mM KCl, and 1 mM CaCl2. The peels were then fixed in 1.5% formaldehyde, 0.5% glutaraldehyde, 0.1 M PIPES, 5 mM EGTA, 2 mM MgCl2, and 0.05% Triton X-100, pH 6.9 for 35 minutes with gentle shaking. The fixed peels were washed in phosphate-buffered saline (PBS) for 30 minutes with three changes of solution. Then they were spread onto microscope slides, blotted to remove excess solution, and freeze-shattered using the methods of Wasteneys, et al., Freeze Shattering: A Simple and Effective Method for Permeabilizing Higher Plant Cell Walls, 188 Journal of Microscopy 51-61 (1997), the teachings of which are hereby incorporated by reference. Briefly, epidermal peels were collected from the abaxial side of Arabidopsis leaves immediately following their detachment and incubated for one hour in a solution containing 5 mM MES-KOH (pH 6.1), 22 mM KCl, and 1 mM CaCl2, Next, the peels were fixed and the fixed peels were washed in phosphate-buffered saline (PBS) for 30 minutes with three changes of solution. The peels were spread onto a microscope slide, blotted to remove excess solution, and then sandwiched with another slide with clamps. The slide-peels-slide sandwich was submerged in liquid nitrogen before being removed and quickly placed between two aluminum blocks which were precooled in liquid nitrogen. The aluminum block was pressed quickly with a thumb until some shattering sound was heard. The slide sandwich was open quickly and a few drops of the fixative was added to the peels. The peels were then transferred to a centrifuge tube and incubated with 1% Triton X-100 for 1-2 hours. The peels were spread on to slides and dried overnight. The peels adhered to slides were incubated with an enzyme mixture followed by incubating with a second enzyme. The peels were permeabilized, incubated, and blocked. The peels were incubated with antibodies to PLD isoforms or their respective pre-immune sera at 4xc2x0 C. overnight, followed by incubation at room temperature. All antibodies were diluted 1:100 in the blocking solution. The slides were rinsed and then incubated for 2 hours with a second antibody (1:50 dilution), which was conjugated to an alkaline phosphatase (Sigma). After rinsing, slides were incubated at room temperature with the phosphatase substrate fast red/naphthol that contained 0.6 mM levamisole to block endogenous AP activity from tissues. The slides were rinsed three times with PBS and sealed for observation and photographing using a microscope.
PLDxcex1 was labeled with the PLDxcex1 antibody and was clearly detectable in guard cells (FIG. 1, photo A). The red color shown in FIG. 1, photo A indicates positive labeling, resulting from the activity of alkaline phosphatase conjugated to a second antibody, whereas labeling with the PLDxcex1 preimmune serum gave negligible background (FIG. 1, photo B). The labeling specificity for PLDxcex1 was verified unequivocally by the absence of immunostaining in guard cells from PLDxcex1-depleted plants (FIG. 1, photo C). Antisense suppression of PLDxcex1 resulted in a nearly complete loss of PLDxcex1 in Arabidopsis leaves, as indicated by the absence of PLDxcex1 activity (FIG. 2) and protein (FIG. 3). For FIGS. 2 and 3, both the activity and immunoblot assays used 2,000xc3x97g supernatant of total leaf extracts. Proteins (10 mg/lane) were separated on 10% SDS-PAGE, and the PLDxcex1 band was marked by an arrow. PLDxcex1 presence in guard cells was confirmed using fluorescence confocal imaging and immuno-gold electron microscopy. These results establish that PLDxcex1 is localized in guard cells and that the expression of PLDxcex1 in guard cells is suppressed in PLDxcex1 antisense plants.
The depletion of PLDxcex1 in guard cells provides a means of assessing the role of PLD in stomatal movement. Stomatal closure was determined by measuring diffusion resistance using a steady-state porometer. Briefly, detached Arabidopsis leaves were floated with the abaxial side downward in a solution containing 5 mM MES-KOH (pH 6.1), 22 mM KCl, and 1 mM CaCl2 for 1 hour under the same light conditions used for growing plants. Leaves then were incubated without or with ABA at indicated concentrations. ABA was made as a 10 mM stock solution in 5% dimethyl sulfoxide (DMSO), and the same amount of DMSO (0.005%) was also added to the control solution in all treatments. Stomatal aperture of detached leaves was measured as diffusion resistance with a steady state porometer using the method of Thimann and S. O. Satler, Relation Between Leaf Senescence and Stomatal Closure: Senescence in Light, 76 Proc. Natl. Acad. Sci. USA, 2295-2298 (1979). For tobacco plants, leaf diffusion resistance was also measured in leaves attached to approximately 2-month-old plants following foliar spraying of ABA at indicated concentrations. Changes in diffusion resistance in response to ABA in both detached and intact leaves were monitored at indicated time intervals. Before drought treatment was imposed, Arabidopsis plants were grown in a greenhouse for 6-8 weeks and watered regularly. Soil water content in each pot was adjusted to approximately the same level before drought treatment. Plants were subjected to drought by withholding irrigation and the soil surface in each pot was covered with plastic wraps to minimize evaporation. Soil moisture in the 0-20 cm soil layer was monitored during drought using a time domain reflectometer. When ABA (10 xcexcM) was sprayed on plants in some treatments, control groups of plants were sprayed with water in the same amount as for the ABA treatment. Leaves were collected at various times of drought treatment, and leaf water potential ("psgr"w) was measured with a thermocouple psychrometer.
Under normal growing conditions, PLDxcex1-deficient and wild-type plants grew comparably. No differences occurred in plant size, development, and reproduction, or the size and density of guard cells on leaves. Incubation of leaves with 10 xcexcM ABA induced stomatal closure, as indicated by an approximately twofold increase in diffusion resistance in wild-type leaves (FIG. 4). The ABA effect persisted for more than 30 minutes in wild-type plants and then decreased. The same ABA treatment had a much smaller effect on stomatal closure in PLDxcex1-suppressed leaves. The ABA-induced increase in diffusion resistance was approximately 50% of that observed in wild-type leaves and returned to the basal level 20 minutes after ABA application. The response to ABA in PLDxcex1-deficient leaves resembled that of the well-characterized, ABA-insensitive mutant abi-1, which is defective in a protein phosphatase 2C involved in ABA signaling in Arabidopsis guard cells. At the range of 0.5-50 xcexcM ABA tested, the PLDxcex1-depleted leaves exhibited a lower diffusion resistance than that of wild-type (FIG. 5). The 2 xcexcM concentration of ABA stimulated stomatal closure in wild-type leaves but had no effect in PLDxcex1-suppressed leaves. The effect in wild-type leaves reached a plateau at 10 [M ABA, whereas such a plateau was not observed at 50 xcexcM ABA in PLDxcex1-suppressed leaves. For each of the graphs in FIGS. 4 and 5, leaves were detached and incubated with the abaxial side down in solutions with different levels of ABA for 20 minutes. Values are meansxc2x1SE of two experiments. These results indicate that PLDxcex1-depleted leaves were less sensitive to ABA.
To determine whether the impaired stomatal closure compromises the plant""s ability to cope with water stress, plants were subjected to progressive drought by withholding irrigation. During drought, PLDxcex1-deficient plants wilted earlier than wild-type plants (FIG. 6). A greater loss of water in leaves was indicated by the lower leaf water potentials in PLDxcex1-deficient plants than in wild-type plants (FIG. 7). By the time 5 days of drought treatment had elapsed, the decrease of water potential was twofold greater in PLDxcex1-deficient than in wild-type leaves. Again, before drought stress was initiated, soil water content in each pot was adjusted to approximately the same level and the soil surface was covered with plastic wrap, so that the water loss from the soil came primarily from leaf transpiration. Measurement of soil water content showed an accelerated decrease with PLDxcex1-deficient plants (FIG. 8), indicating a greater transpirational loss of water in these plants.
Additionally, ABA (10 xcexcM) was sprayed on a set of drought-stressed plants once a day to test its effect on promoting drought resistance in PLDxcex1-depleted and wild-type plants. This treatment enhanced resistance to drought in wild-type plants, as indicated by the maintenance of leaf turgidity during drought (FIG. 6) increased leaf water potential (FIG. 7), and soil water content (FIG. 8). The same ABA treatment had no detectable effect on water loss and drought resistance of PLDxcex1-deficient plants. These data provide in planta evidence that suppression of PLDxcex1 decreased plant sensitivity to ABA. This reduction in ABA-induced stomatal closure resulted in increased transpirational water loss in PLDxcex1-deficient plants.
To verify the role of PLD in stomatal closure, PLDxcex1-overexpressing tobacco was used to determine the effect of increased PLDxcex1 expression on the rate of water loss and ABA-induced stomatal closure. FIGS. 9-12 illustrate these results showing increased sensitivity to ABA-promoted stomatal closure and decreased water loss in PLDxcex1-overexpressing tobacco. Introduction of a PLDxcex1 construct to tobacco resulted in approximately a fivefold increase in PLDxcex1 activity (FIG. 9). Expression of the introduced PLDxcex1 was attested clearly by the presence of a protein band of slightly smaller molecular weight than the tobacco endogenous PLD (FIG. 9, inset). For this immunoblot, proteins (10 mg/lane) were separated on 10% SDS-PAGE, and PLDxcex1 was made visible by staining with alkaline phosphatase. The arrow marks the overexpressed PLDxcex1. Both the activity and immunoblot assays used 2,000xc3x97g supernatant of total leaf extracts. The introduced PLDxcex1 was expressed in tobacco guard cells. Multiple PLDxcex1-overexpressing lines have been produced, and all grew and developed normally to maturity. Cellular fractionation showed that the introduced PLDxcex1 had the same intracellular association as the endogenous PLDxcex1, being present in both soluble and microsomal membrane fractions. The PLDxcex1-elevated and wild-type plants also showed no significant differences in leaf phospholipid content and composition (data not shown). Moreover, these observations indicate that PLDxcex1 activity is tightly regulated after translation.
The large size of tobacco leaves permitted measurement of transpirational water loss directly on plants after ABA treatments. As shown in FIG. 10, when leaves were sprayed with 2.5 and 5 xcexcM ABA, stomata closed faster and more tightly in the PLDxcex1-overexpressing than in control plants (tobacco transformed with an empty vector). Leaf diffusion resistance was measured directly on plants that were sprayed with ABA and expressed as percentages of that of plants sprayed with water. Leaf diffusion resistance increased about 80% in PLDxcex1-overexpressing plants while diffusion resistance in leaves of control plants increased only about 30% 20 minutes after ABA application. As shown in FIG. 10, the differences in diffusion resistance between the two genotypes were most noticeable within the first 20 minutes after ABA application and diminished afterwards. These differences indicate that overexpression of PLDxcex1 enhances plant sensitivity to ABA and also implies that PLD activation could be a limiting step in the early stages of ABA induced stomatal movement induced by ABA.
To assess water loss from leaves without added ABA, leaves of similar size, age, and positions on PLDxcex1-overexpressing and control plants were detached and measured for decreases in fresh weight. Leaves from PLDxcex1-overexpressing plants exhibited markedly lower rates of water loss than those from control plants under ambient conditions (FIG. 11). The differences occurred within 5 minutes and became more apparent between 20 to 30 minutes following detachment. Values are percentages of the meansxc2x1SE of three experiments. These results show that overexpression of PLDxcex1 promotes stomatal closure induced by ABA and/or water deficit and decreases transpirational water loss.
To further demonstrate the effects of PLDxcex1 overexpression, PLDxcex1 overexpressing tobacco plants and empty vector-transformed tobacco plants were compared. Six week-old tobacco plants of similar sizes were subjected to drought by withholding irrigation for 15 days in a growth room with cool-white fluorescent lights at 23xc2x12xc2x0 C. and 45% relative humidity. As shown in FIG. 12, the PLDxcex1 overexpressing plants exhibited increased resistance to drought through increased turgidity.
With PLDxcex1-depleted Arabidopsis and PLDxcex1-overexpressing tobacco, the present invention illustrates that PLD constitutes a critical step in ABA signaling and plant response to water stress. A look at the biochemical and cellular properties of PLD may indicate PLD""s role in mediating ABA action in stomatal closing. Increasing cytoplasmic Ca2+ oscillation is a key step in the ABA signal transduction. Mutation or inhibition of the ABA signaling components, such as protein phosphatase 2C, cADP ribose, protein farnesylation, and phospholipase C, impedes ABA-induced Ca2+ oscillation and impairs stomatal closure. Ca2+ is a regulator of plant PLD in that it is required for PLD activity and it also promotes PLDxcex1 association with membranes as shown by S. B. Ryu and X. Wang, Activation of Phospholipase D and the Possible Mechanism of Activation in Wound-Induced Lipid Hydrolysis in Castor Bean Leaves, 1303 Biochimica et Biophysica Acta, 243-250 (1996), the methods and teachings of which are hereby incorporated by reference. PLD binds Ca2+ at its N-terminal C2 domain, thereby inducing a conformational change and promoting the protein association with phospholipids. ABA exposure increases PLD activity in guard cells as shown by Zheng, et al., Distinct Ca2+ Binding Properties of Novel C2 Domains of Plant Phospholipase Da and xcex2, 275 The Journal of Biological Chemistry 19700-19706 (2000), the methods and teachings of which are hereby incorporated by reference. Thus, PLD could be a target of Ca2+ oscillation that activates PLD in guard cells.
PLD activation generates the lipid product PA which, when applied to guard cell protoplasts, results in an increase in ionic efflux. Although the mechanism by which PA mediates cellular effect is unknown in plants, PLD-derived PA can activate protein kinases and lipid kinases in animal systems. In particular, PA is a potent stimulator of phosphatidylinositol 5-kinases for the production of phosphatidylinosotol 4,5-bisphosphate, which is a substrate for PI-PLC and also is essential for membrane trafficking and cytoskeletal dynamics. Active membrane trafficking and cytoskeletal rearrangements have been implicated in stomatal movement. In addition, PA may carry out its cellular effect via membrane structural alteration. It is a nonlamellar lipid and favors the formation of hexagonal phase II in the presence of calcium. The formation of PA and lysoPA occurs specifically at the neck of a budding synaptic vesicle and is required in membrane budding.
Thus, the present invention also includes methods of creating transformed plants by recombinantly altering the genome of the plants such that their PLDxcex1 expression is altered when compared to a baseline level of PLDxcex1 expression in wild type plants. To determine whether or not the genome alteration has effected stomatal closure characteristics, such characteristics are determined. In some instances, the genome alteration results in an up-regulation of PLDxcex1 expression and in other cases results in a down-regulation of PLDxcex1 expression. A preferred method of up-regulating PLDxcex1 expression includes introducing an insert which codes for PLDxcex1. Preferably the insert includes a promoter and PLDxcex1 encoding sequences. The preferred PLDxcex1 coding sequence is included herein as SEQ ID No. 2. Preferably, sequences having at least about 60% sequence similarity or 50% sequence identity to SEQ ID No. 2 are used to up-regulate PLDxcex1 expression by being introduced into the PLD genome. More preferably, such sequences have at least about 70% sequence similarity or 65% sequence identity with SEQ ID No. 2. Most preferably, such sequences have at least about 90% sequence similarity or 85% sequence identity with SEQ ID No. 2. The promoter used is preferably a constitutive promoter and a particularly preferred promoter used to control the inserted sequence is the 35S promoter from the cauliflower mosaic virus. Of course, other promoters such as the ubiquitin promoter would reasonably be expected to work in a similar fashion for purposes of the present invention. These types of promoters provide high levels of expression of heterologous genes in a variety of different cell and tissue types of many dicot and monocot plant species.
Stomatal closure characteristics can be tested in a variety of ways. For example, the transpiration rate of plants can be tested as can the plant""s diffusion resistance. Additionally, testing conditions can be varied such that the conditions under which the plants are grown are not conducive to the growth of unmodified or untransformed plants. A preferred testing condition includes subjecting the plants to drought conditions or excessive water conditions. Another form of testing stomatal closure characteristics includes observing the turgidity of plants. This type of observation provides an easily observable phenotypic trait of plants which is directly related to stomatal closure.
The present invention also provides methods of growing transformed plants in locations having unsuitable water and growth conditions for untransformed plants. These methods generally include the steps of recombinantly altering the genome of the plant in an effort to change the level or amount of PLD expressed by the plant, testing water consumption levels of the plant in order to determine if the genome alterations permit plant growth in the unsuitable locations, and planting the progeny of the plant in the conditions which were unsuitable for growth of untransformed plants. Again, preferred alterations are similar to the ones described above for testing the stomatal closure characteristics of plants after genome alteration. By testing the water consumption level of the plant, it will become apparent whether or not the resulting plant and its progeny will be adapted to live in either an environment which has too much soil moisture or, alternatively, too little soil moisture to support normal plant growth. The plant progeny which are adapted for and conditions, as a result of the alteration of the genome, will grow in areas which were previously too dry for plants with unaltered genomes. Conversely, plant progeny which have altered genomes which have increased levels of water consumption (and transpiration) will be able to grow in environments which had previously had too much moisture in the soil to support the growth of plants with unmodified or unaltered genomes. Testing methods for growth of progeny will include testing transpiration rate, diffusion resistance, effects of abscisic acid exposure, effects of drought conditions, and effects of overly wet conditions. Phenotypic testing methods will include observing the plant""s turgidity.
Finally, the present invention provides methods of growing transformed plants which have modified stomatal closure responses to water availability in comparison to untransformed plants. Untransformed plants exhibit a baseline stomatal closure response while plants which have been successfully transformed, have stomatal closure responses which differ from that baseline. For the transformed plants, the genome is recombinantly altered in an effort to change the stomatal closure responses and the resultant plants are tested for their stomatal closure responses and then compared to those of untransformed plants to determine whether or not the transformed plant has modified stomatal closure responses. Again, it is preferred to use the same experimental and modifications to these plants as previously described.
In conclusion, the present results demonstrate that PLD plays a crucial role in plant transpiration. Through targeted manipulation of the specific PLD sequence in guard cells permits generation of plants with decreased water consumption and enhanced tolerance to water stress. Alternatively, manipulation of PLD expression may promote increased rates of plant transpiration and be more efficient in bioremediation.